Regulation of Axial Extension in Vertebrate Embryos

脊椎动物胚胎轴向延伸的调节

• 批准号: 8519476
• 负责人: Paul Michael Skoglund
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8519476
• 关键词: Actins; Acute; Adhesions; Anencephaly; Anterior; Automobile Driving; Biological; Biological Assay; Biomechanics; Blastopores; Cadherins; Cell Shape; Cell-Cell Adhesion; Cells; Chemicals; Complex; Congenital Abnormality; Crosslinker; Data; Defect; Dependence; Development; Diagnostic; Dorsal; Embryo; Equilibrium; Event; Failure; Feedback; Filament; Germ Layers; Goals; Health; Homeostasis; Human; Infertility; Intercalated Cell; Lead; Lip structure; MYLK gene; Mediating; Mesoderm Cell; Modeling; Molecular; Morphogenesis; Motor; Myosin ATPase; Myosin Regulatory Light Chains; Myosin Type II; Neural Tube Closure; Neurons; Nonmuscle Myosin Type IIB; Pathway interactions; Pattern; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Probability; Process; Production; Proteins; Rana; Regulation; Research Personnel; Role; Series; Shapes; Signal Transduction; Spinal Dysraphism; Stress; Stretching; System; Takeda brand of pioglitazone hydrochloride; Testing; Therapeutic Intervention; Tissues; Up-Regulation; Western Blotting; Work; base; crosslink; gastrulation; inhibitor/antagonist; intercalation; metaplastic cell transformation; myosin VI; myosin phosphatase; non-muscle myosin; novel; relating to nervous system; research study

DESCRIPTION (provided by applicant): The proposal "Regulation of Vertebrate Axial Extension" aims to expand on the observation previously made by this investigator that a cortical acto-myosin network is required for the cellular convergence and extension that drives the shape change of frog embryos at gastrulation. Because this sub-membranous actin network requires the myosin IIB (MIIB) complex to function in this morphogenesis we aim to determine whether this MII dependence requires actin-activated contractility, or if MII functions solely as a actin crosslinker during axial extension. Preliminary evidence depleting the myosin regulatory light chain (MRLC), required for expression of contractile but not crosslinking activity in MII, indicates that myosin II contractility is required. Because regulation of MII contractility is controlled by the phosphorylation state of Ser19 on MRLC, we perturbed a possible dynamic homeostasis of this Ser19-P event by adding an inhibitor of myosin phosphatase to embryos, and assaying for Ser19 phosphorylation by quantitative western blotting to reveal an acute upregulation of Ser19-P. This experiment reveals that there is an active, balanced process involving myosin phosphatase and a kinase or kinases to regulate Ser19-P levels in intercalating cells, and further acute drug experiments indicate that the relevant kinase is MLCK. Because Ca ++ is an upstream regulator of MLCK, we reexamined Ca++ dynamics in intercalating cells, finding a novel pattern of Ca++ fluxes, as well as a plausible mechanism for generating these fluxes through a putative Ca++ channel protein that we show to be required for gastrulation. We also identify a role for a second motor protein in regulating the cortical actin network, allowing us to propose a model for CE consisting of a simple oscillating regulatory circuit with biomechanical feedback control, as well as the means to test this hypothesis.

描述（由申请人提供）：“脊椎动物轴向伸展的调节”提案旨在扩展本研究者先前的观察结果，即细胞会聚和伸展需要皮质肌动蛋白-肌球蛋白网络，该网络驱动青蛙胚胎在原肠胚形成时的形状变化。由于这种膜下肌动蛋白网络需要肌球蛋白IIB（MIIB）复合物在这种形态发生中发挥作用，我们的目标是确定这种MII依赖是否需要肌动蛋白激活的收缩性，或者MII是否仅在轴向延伸过程中作为肌动蛋白交联剂发挥作用。初步证据耗尽肌球蛋白调节轻链（MRLC），所需的表达收缩，但不是交联活性的MII，表明肌球蛋白II收缩性是必需的。由于MII收缩性的调节受MRLC上Ser 19的磷酸化状态控制，我们通过向胚胎中加入肌球蛋白磷酸酶抑制剂来扰乱该Ser 19-P事件的可能的动态稳态，并通过定量蛋白质印迹分析Ser 19磷酸化以揭示Ser 19-P的急性上调。肌球蛋白磷酸酶和一种或多种激酶参与的调节嵌入细胞中Ser 19-P水平的平衡过程，进一步的急性药物实验表明相关激酶是MLCK。由于Ca ++是MLCK的上游调节因子，我们重新研究了嵌入细胞中的Ca++动力学，发现了一种新的Ca++通量模式，以及通过一种假定的Ca++通道蛋白产生这些通量的合理机制，我们表明这是原肠胚形成所需的。我们还确定了第二个电机蛋白在调节皮层肌动蛋白网络的作用，使我们能够提出一个模型CE组成的一个简单的振荡调节电路与生物力学反馈控制，以及测试这一假设的手段。